CA2184386C - Interference eliminating agent for application in immunoassays - Google Patents
Interference eliminating agent for application in immunoassays Download PDFInfo
- Publication number
- CA2184386C CA2184386C CA002184386A CA2184386A CA2184386C CA 2184386 C CA2184386 C CA 2184386C CA 002184386 A CA002184386 A CA 002184386A CA 2184386 A CA2184386 A CA 2184386A CA 2184386 C CA2184386 C CA 2184386C
- Authority
- CA
- Canada
- Prior art keywords
- streptavidin
- avidin
- derivative
- biotin
- analyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000003018 immunoassay Methods 0.000 title claims abstract description 26
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 127
- 108090001008 Avidin Proteins 0.000 claims abstract description 94
- 230000003993 interaction Effects 0.000 claims abstract description 14
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 126
- 239000011616 biotin Substances 0.000 claims description 65
- 229960002685 biotin Drugs 0.000 claims description 65
- 235000020958 biotin Nutrition 0.000 claims description 62
- 238000009739 binding Methods 0.000 claims description 50
- 230000027455 binding Effects 0.000 claims description 48
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- NGXDNMNOQDVTRL-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(4-azido-2-nitroanilino)hexanoate Chemical compound [O-][N+](=O)C1=CC(N=[N+]=[N-])=CC=C1NCCCCCC(=O)ON1C(=O)CCC1=O NGXDNMNOQDVTRL-UHFFFAOYSA-N 0.000 description 2
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- FRTGEIHSCHXMTI-UHFFFAOYSA-N dimethyl octanediimidate Chemical compound COC(=N)CCCCCCC(=N)OC FRTGEIHSCHXMTI-UHFFFAOYSA-N 0.000 description 1
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- 125000001997 phenyl group Chemical class [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/824—Immunological separation techniques
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/825—Pretreatment for removal of interfering factors from sample
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25125—Digestion or removing interfering materials
Abstract
The invention concerns interference-eliminating agents for avoiding unspecific interactions in immunoassays in which avidin or streptavidin or a derivative thereof are used as the interference-eliminating agents.
Description
218438b Interference eliminating agent for application in immunoassays The invention concerns avidin or streptavidin or a derivative thereof as an interference-eliminating agent in immunoassays as well as methods for the detection of an analyte using this interference-eliminating agent.
Immunological methods of detection have become of great importance in recent years. They can be used to rapidly and accurately detect the presence of drugs, hormones, proteins, infectious organisms and in particular specific antibodies in biological samples. In all immunological methods of detection a specific binding reaction occurs between a first specific binding partner, the substance which it is intended to detect ("analyte") and a second specific binding partner which reacts specifically with the analyte or binds it. In this process the analyte and specific analyte binding partner, the so-called partners of a specific binding pair, form a specific binding pair which is in general a complex between an antigen and an antibody or antibody fragment. Tn this connection it is possible for more than one analyte or binding partner to react with one another in each reaction. These specific binding reactions are detected in various ways. In general one participant in the specific binding reaction is labelled. Common labelling methods are radioisotopes, chromogens, fluorogens, enzyme labels or substances which in turn can form a specific binding pair (e. g.
biotin/streptavidin). In heterogeneous immunoassays one of the binding partners is immobilized on a solid phase.
Immunological methods of detection have become of great importance in recent years. They can be used to rapidly and accurately detect the presence of drugs, hormones, proteins, infectious organisms and in particular specific antibodies in biological samples. In all immunological methods of detection a specific binding reaction occurs between a first specific binding partner, the substance which it is intended to detect ("analyte") and a second specific binding partner which reacts specifically with the analyte or binds it. In this process the analyte and specific analyte binding partner, the so-called partners of a specific binding pair, form a specific binding pair which is in general a complex between an antigen and an antibody or antibody fragment. Tn this connection it is possible for more than one analyte or binding partner to react with one another in each reaction. These specific binding reactions are detected in various ways. In general one participant in the specific binding reaction is labelled. Common labelling methods are radioisotopes, chromogens, fluorogens, enzyme labels or substances which in turn can form a specific binding pair (e. g.
biotin/streptavidin). In heterogeneous immunoassays one of the binding partners is immobilized on a solid phase.
A serious problem in immunoassays is that undesired interactions and unspecific binding reactions can occur between specific binding partners of the immunoassay and -the sample, additional constituents present in the sample and under certain circumstances in the solid phase. Such interactions usually cause an increase of the background signal and also a larger scattering of the signals and thus a reduced sensitivity and specificity of the test concerned. False-positive measurements can also result from an unspecific interaction with the labelled binding partner as well-as-from the specific binding of test components by sample constituents i.e. as a result of the falsely-increased measured signal it is assumed that an analyte is present even when it is absent.
Many attempts have been made to reduce these unspecific interactions in immunoassays. It has been known for a long time that various carbohydrate components and various proteins, protein mixtures or protein fractions as well as hydrolysates thereof can reduce unspecific interactions between the test components and the analyte in immunoassays (for example Robertson et al., Journal of Immun. Meth. 26, 1985, 195; EP-A-260903; US-A-4,931,385). The use of crude protein fractions and crude hydrolysates has the disadvantage that the constituents contained therein can in turn cause other interferences of the test. Moreover hydrolysates produced by enzymatic means may be contaminated with the proteases used for their manufacture and usually do not have a uniform quality since the cleavage is difficult to control.
Protease contaminations can attack test components and lead to an impairment of the test functions and storage stability even in low amounts.
Many attempts have been made to reduce these unspecific interactions in immunoassays. It has been known for a long time that various carbohydrate components and various proteins, protein mixtures or protein fractions as well as hydrolysates thereof can reduce unspecific interactions between the test components and the analyte in immunoassays (for example Robertson et al., Journal of Immun. Meth. 26, 1985, 195; EP-A-260903; US-A-4,931,385). The use of crude protein fractions and crude hydrolysates has the disadvantage that the constituents contained therein can in turn cause other interferences of the test. Moreover hydrolysates produced by enzymatic means may be contaminated with the proteases used for their manufacture and usually do not have a uniform quality since the cleavage is difficult to control.
Protease contaminations can attack test components and lead to an impairment of the test functions and storage stability even in low amounts.
The use of chemically modified proteins, in particular of succinylated or acetylated proteins, has also been described for the reduction of unspecific interactions in immunoassays (US-A-5,051,356; EP-A-0 525 916).
However, i.t was not possible to avoid many of the false positive results in tests for antibodies from serum with these substances.
EP-A-0 331 068 and WO 91/06559- describe the use of polymerized immunoglobulins, in particular IgG, to reduce specific interfering factors such as e.g.
rheumatoid factors. However, they do not enable all interfering interactions to be satisfactorily eliminated. Moreover, the addition of unspecific human immunoglobulin (monomeric or polymeric antibodies or fragments thereof) in tests for human antibodies can lead to an increase in the blank. Furthermore the production of human or animal IgG is time-consuming and expensive.
The object of the invention was therefore to provide new interference-eliminating substances and interference-eliminating agents which reduce interference by unspecific interactions in immunoassays which is better than that known from the state of the art. Unspecific interactions are understood as all interactions between components of the process which can lead to falsifications of the measured result. The interference-eliminating substances should avoid false-positive analytical results in particular in the analysis of antibodies. In particular it is intended to avoid interference when using avidin or streptavidin as a binding partner in an immunoassay.
~
However, i.t was not possible to avoid many of the false positive results in tests for antibodies from serum with these substances.
EP-A-0 331 068 and WO 91/06559- describe the use of polymerized immunoglobulins, in particular IgG, to reduce specific interfering factors such as e.g.
rheumatoid factors. However, they do not enable all interfering interactions to be satisfactorily eliminated. Moreover, the addition of unspecific human immunoglobulin (monomeric or polymeric antibodies or fragments thereof) in tests for human antibodies can lead to an increase in the blank. Furthermore the production of human or animal IgG is time-consuming and expensive.
The object of the invention was therefore to provide new interference-eliminating substances and interference-eliminating agents which reduce interference by unspecific interactions in immunoassays which is better than that known from the state of the art. Unspecific interactions are understood as all interactions between components of the process which can lead to falsifications of the measured result. The interference-eliminating substances should avoid false-positive analytical results in particular in the analysis of antibodies. In particular it is intended to avoid interference when using avidin or streptavidin as a binding partner in an immunoassay.
~
This object was achieved by avidin or streptavidin or derivatives thereof as interference-eliminating agents.
The use of these substances surprisingly resulted in an elimination of interference in assays in particular in assays in which avidin or streptavidin is used as a binding partner.
The invention therefore concerns interference-eliminating agents to avoid unspecific interactions in assays which contain avidin or streptavidin or derivatives thereof and the use of these interference-eliminating agents in assays. Avidin or streptavidin or derivatives thereof are used in a soluble form according to the invention. They are not bound or coupled to a solid phase or a marker group such as an enzyme. The interference-eliminating agent according to the invention is always used in addition to the reagents that are otherwise necessary for-the test and does not become a component of the complex which forms during the course of the test reaction composed of analyte to be detected and specific binding partners. The interference-eliminating agent according to the invention is preferably used in immunological tests (immunoassays). It can, however, also be used in tests which are based on other interactions such as nucleic acid tests. The interference-eliminating agent is preferably used in tests in which one test component is bound to avidin or streptavidin such as an avidin-coated or streptavidin-coated solid phase in the case of -immunological or nucleic acid tests. Avidin or streptavidin are understood as the naturally occurring purified proteins or recombinant avidin or streptavidin.
Avidin or streptavidin derivatives are understood as modified avidin or streptavidin molecules. The modification can firstly be achieved by cross-linking individual avidin or streptavidin molecules so that so-called poly SA or homogeneously cross-linked SA is formed. SA in the following is always understood as avidin or streptavidin. Methods for cross-linking or polymerizing SA are known to a person skilled in the art. In particular a cross-linking by heat treatment or -.
by bifunctional or polyfunctional compounds is suitable.
Bifunctional or polyfunctional compounds are understood as molecules which carry at least two functional groups which can be the same or different and can react via these functional groups with functional groups of SA
such as certain amino acid residues or carbohydrate residues. Typical examples of linkers suitable within the scope of the invention are listed in the following table 1.
The use of these substances surprisingly resulted in an elimination of interference in assays in particular in assays in which avidin or streptavidin is used as a binding partner.
The invention therefore concerns interference-eliminating agents to avoid unspecific interactions in assays which contain avidin or streptavidin or derivatives thereof and the use of these interference-eliminating agents in assays. Avidin or streptavidin or derivatives thereof are used in a soluble form according to the invention. They are not bound or coupled to a solid phase or a marker group such as an enzyme. The interference-eliminating agent according to the invention is always used in addition to the reagents that are otherwise necessary for-the test and does not become a component of the complex which forms during the course of the test reaction composed of analyte to be detected and specific binding partners. The interference-eliminating agent according to the invention is preferably used in immunological tests (immunoassays). It can, however, also be used in tests which are based on other interactions such as nucleic acid tests. The interference-eliminating agent is preferably used in tests in which one test component is bound to avidin or streptavidin such as an avidin-coated or streptavidin-coated solid phase in the case of -immunological or nucleic acid tests. Avidin or streptavidin are understood as the naturally occurring purified proteins or recombinant avidin or streptavidin.
Avidin or streptavidin derivatives are understood as modified avidin or streptavidin molecules. The modification can firstly be achieved by cross-linking individual avidin or streptavidin molecules so that so-called poly SA or homogeneously cross-linked SA is formed. SA in the following is always understood as avidin or streptavidin. Methods for cross-linking or polymerizing SA are known to a person skilled in the art. In particular a cross-linking by heat treatment or -.
by bifunctional or polyfunctional compounds is suitable.
Bifunctional or polyfunctional compounds are understood as molecules which carry at least two functional groups which can be the same or different and can react via these functional groups with functional groups of SA
such as certain amino acid residues or carbohydrate residues. Typical examples of linkers suitable within the scope of the invention are listed in the following table 1.
Table 1 Abbreviation Chemical composition SPDP N-succinimidyl-3-(2-pyridyldithio)-propionate EADP ethyl 4-azidohenyl-1,4-dithiobutyrimidate HC1 FNPA 4-fluoro-3-nitrophenylazide HSAB N-hydroxysuccinimidyl-4-azidobenzoate MABI methyl-4-azidobenzoimidate HC1 MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester NHS-ASA N-hydroxysuccinimidyl-4-azidosalicylic acid MHS maleimidohexanoyl-N-hydroxysuccinimide ester PNP-DTP p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate SADP N-succinimidyl(4-azidophenyl)1,3'-dithiopropionate SAND sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopropionate SANPAH N-succinimidyl-6(4'-azido-2'-nitrophenyl-amino)hexanoate SASD sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate SMCC succinimidyl-4-(N-maleinimidoethyl)cyclohexane-1-carboxylate SMPB succinimidyl-4-(p-malainimidophenyl)butyrate DSS disuccinimidylsuberate DMS dimethylsuberimidate Traut' reagent 2-iminothiolane 2,4,6-trichloro-s-triazine SAMBA S'-acetyl-mercapto-succfnic acid anhydride SATP N-succinimidyl-S-acetylthiopropionate -SATA N-succinimidyl-S-acetylthioacetate Secondly the modification can be a coupling to other high molecular molecules in particular proteins such as bovine serum albumin (BSA) or immunoglobulin which are -.
often used to reduce interference in immunoassays. This cross-linking leads to so-called heterogeneously cross-linked SA. In this process SA and/or the protein can in turn be polymerized in this complex. Methods for coupling SA to these molecules are known to a person -killed in the art. A coupling with DSS, SAMBA, SATP, MAS, SATA has proven to be particularly suitable. A
complex of BSA and SA or poly SA is particularly suitable as an interference-eliminating agent. Even better effects are achieved when polymerized BSA is coupled to SA or poly SA. The polymerization or cross-linking of BSA can be achieved with the aforementioned methods for cross-linking SA. The most suitable is a polymerization by heat treatment as described in EP-A
0331 127.
Thirdly the modification of SA can be an inactivation of the active centres i.e. the binding sites forbiotin.
This results in the formation of so-called inactivated SA. The inactivation can be achieved by a simple saturation of the binding sites with biotin or with a biotin derivative which is bound by avidin or streptavidin. Due to the very high affinity of SA for biotin hardly any biotin which could eventually lead to interferences is released when used in the immunoassay.
The active centre is preferably covalently modified. In this case one or several amino acid residues in the active centre of SA can be derivatized so that binding to biotin is greatly reduced or completely prevented.
Methods for inactivating the biotin binding site in SA
are known from Gitlin et al., Biochem. J. 269 (1990) _$_ 527-530. Tyrosine residues in the active centre of SA
are preferably modified with p-nitrobenzenesulfonyl fluoride. A further method for inactivating the biotin binding site in SA is to covalently couple biotin to the binding site. Methods for coupling biotin to the active centre of SA are known to a person skilled in the art. A
new method for coupling biotin to SA by means of a new photoactivatable biotin derivative has turned out to be particularly preferable. Photoactivatable biotin derivatives are known. In EP-A-0 155 854 and EP-A-0 187 323 azide-substituted phenyls/nitrophenyls are described which are coupled by an amine-containing linker to biotin. Biotin-DAD00-AB (biotin-[8-(4-azidobenzoyl)-amino-3,6-dioxaoctyl]amide) is preferably used as the biotin derivative. Further biotin derivatives are described in the Boehringer Mannheim Biochemica catalogue order no. 1292633 and 1292641. After saturation of the biotin binding sites of SA with the biotin derivatives, the photoreaction is initiated and thus biotin is covalently fixed to the active centre. In this inactivated SA biotin is bound to the active centre and additionally bound outside the active centre via a covalent bond. The SA biotin product obtainable by this means is also a subject matter of the present invention.
A further method of inactivating the active centre of SA
is to modify the binding sites by genetic engineering.
The active centre of SA can be modified and inactivated by substitution, deletion or insertion of individual amino acid residues or short sections of amino acid residues. Preferably individual amino acids such as tyrosine residues are substituted by other amino acids in the active centre. It is, however, also possible to produce SA in which the binding site is partially or completely absent. Methods for modifying and producing SA by means of genetic engineering are known to a person skilled in the art. For example the production of recombinant streptavidin is described in EP-A-0 198 015.
Fourthly the modification can be achieved by a fragmentation of SA. The SA fragments can be produced by chemical or enzymatic cleavage or by recombinant production. In-this case the biotin binding site in particular may be absent as already described above. The advantage of fragmentation is an improved solubility of the product. Preferably all fragments obtained by chemical or enzymatic cleavage from avidin or streptavidin are used in the mixture so that all or nearly all parts of SA are present.
The SA inactivated or fragmented in this way can be used in the aforementioned first or second method for modifying SA i.e. the inactivated SA can in turn be polymerized or cross-linked with other molecules such as BSA or poly BSA. These interference-eliminating agents in which a combination of at least two of the said methods have been carried out to modify the SA have proven to be particularly advantageous.
Afterthe modification of SA by one of the said methods has been completed it is possible to remove remaining biotin binding activity, biotin covalently bound to the surface which is not bound in the active centre of -avidin or streptavidin or free biotin by a suitable purification of the avidin or streptavidin derivatives.
This can for example be achieved by adsorption to solid phase bound biotin and/or SA.
The invention therefore in addition concerns avidin or - to - 218~38~
streptavidin derivatives obtainable by one of the methods described above for modification and subsequent purification to remove remaining biotin binding activity, biotin covalently bound to the surface which is not bound in the active centre of avidin or streptavidin or free biotin preferably by adsorption to a solid phase bound biotin and/or SA.
Furthermore the invention concerns a process for the production of the interference-eliminating agents -according to the invention in which SA is modified according to at least one of the three aforementioned methods and is optionally subsequently purified as described above to remove remaining biotin binding capacity, biotin bound covalently to the surface or free biotin.
Avidin or streptavidin or derivatives thereof can be used to reduce interference in all common immunoassays or nucleic acid assays. They are particularly suitable for reducing interference in immunoassays or nucleic acid assays in which avidin or streptavidin is used as a binding component. Such immunoassays are for example known from Guesdon et al., J. Histochem. Cytochem. 27 (1979) 1131-1139 and Bayer and Wilchek, Analytical Biochemistry 171 (1988) 1-32. The interferences can for example be caused by antibodies against avidin or streptavidin which sometimes occur in human serum.
However, the interference-eliminating agent according to the invention also exhibits an advantageous effect in immunoassays in which no avidin or streptavidin is used.
In these immunoassays the use of SA which is bound to a further molecule such as for example BSA or poly BSA
corresponding to the second method of modification described above has proven to be particularly advantageous in these immunoassays. The interference- _ eliminating agent according to the invention is always used in addition to the reagents that are otherwise necessary in the test. It is not identical to the avidin or streptavidin components which may possibly be present in the test such as an SA-coated solid phase or enzyme-SA conjugates. In contrast to these SA reagents the interference-eliminating reagent according to the invention is not incorporated into the complex to be detected composed of analyte and specific binding partners.
The invention furthermore concerns a method for the determination of an analyte in a sample by (1) contacting the sample with (a) avidin or streptavidin or a derivative thereof (b) one or several specific binding partners of the analyte and (2) measuring the complex formed from analyte and specific binding partners as a measure for the presence of the analyte.
All substances can serve as the analyte which react to form a complex with at least one specific binding partner such as for example haptens, antigens, antibodies or nucleic acids. The method according to the invention is particularly suitable for the detection of antibodies in particular autoantibodies.
In general body fluids such as blood, plasma, serum, -1z-saliva or urine serve as the sample.
Any biological or chemical binding partners can serve as the specific binding partner which is capable of specifically binding the analyte and can form a complex with it. These include antibodies, antibody fragments, antigens, haptens, hormones, avidin, biotin, nucleic acids, oligonucleotides or derivatives thereof.
Antibodies or antigens or fragments thereof are preferably used in the present invention as a binding partner of the analyte.
In order to detect the complex composed of analyte and specific binding partner it is possible to use all methods familiar to a person skilled in the art. It is possible to use homogeneous methods in which all binding partners in the method are in a soluble form such as precipitation methods with a turbidimetric or nephelometric determination of the complex formed or immunoassays based on the CEDIA, EMIT or FPIA principle.
Heterogeneous methods are also suitable in which at least one reagent is bound to a solid phase. Examples of this are agglutination tests in which one partner of a binding pair is for example bound to latex, sandwich assays, ELISA for the detection of specific antibodies such as e.g. against HIV, HCV, rubella, toxoplasma gondii, glutamate decarboxylase or thyroglobulin, RIA or immunometric assays. Apart from the precipitation methods, one of the specific binding partners is labelled in all these methods. The label can directly generate a measurable signal which is for example a radioisotope, a chemiluminescent, fluorescent or electrochemiluminescent label or a coloured particle such as a metal sol particle or dyed or undyed latex.
The label can also generate an indirect signal such as an enzyme label like peroxidase, glucose oxidase, f3-galactosidase or alkaline phosphatase.
The immunoassays can also be carried out by means of test strips or biosensors in particular when one of the binding partners is coupled via SA to the solid phase.
Interference in immunoassays based on the principle plasmon resonance can also be reduced according to the invention.
One reagent of the method for detecting the analyte is often coupled via a specific binding pair such avidin or streptavidin/biotin to the solid-phase. The advantage of this is that the solid phase can be used universally in several test procedures. It is also possible to bind the ._.
label to a component of the assay via a specific binding pair. For example an enzyme can be coupled to avidin or streptavidin and the binding partner, for example an antibody, can be biotinylated. Examples of such test procedures are known to a person skilled in the art. The avidin or streptavidin derivative according to the invention is particularly suitable for reducing interference in those methods of detection which utilize an indirect binding of a reaction component by avidin/
biotin or streptavidin/biotin.
The method for detecting the analyte can be carried out in one or several steps i.e. the incubation of the analyte with the individual test components can be carried out simultaneously or successively. If avidin or streptavidin or a derivative thereof is used in which the biotin binding sites have not been inactivated, then in methods in which a binding component is coupled via avidin/biotin or streptavidin/biotin care must be taken i that binding of the binding components via avidin/biotin or streptavidin/biotin is already completed before avidin or streptavidin or a derivative thereof is added since otherwise the non-inactivated biotin binding sites would bind to the biotinylated binding component and cause interferences. For example when using an avidin or streptavidin solid phase, the biotinylated specific binding partner of the analyte must be added first before the sample is added together with avidin or streptavidin or a derivative thereof. If an avidin or streptavidin derivative according to the invention is used in which the biotin binding sites have been inactivated then all test reagents can be incubated simultaneously since the biotinylated reagents do not bind to the avidin or streptavidin derivative.- This inactivated avidin or streptavidin derivative can thus be used universally in all conceivable test variants and is thus preferred.
The concentration of the interference-eliminating agent according to the invention in the test mixture is -between 0.0001 and 1 ~ (m/v) preferably between 0.01 and 1 ~ (m/v).
The individual reaction components of the method for detecting the analyte are advantageously offered in the form of a test combination or a test kit. Therefore a further subject matter of the invention is a test combination for a method for detecting an analyte in a sample containing avidin or streptavidin or a derivative thereof and at least one specific binding partner of the analyte. In addition it can also contain all other reagents necessary to carry out the test procedure such as buffers, detergents, labels, auxiliary substances to detect the label such as enzyme substrates, solid phases 218438b etc.. The interference-eliminating agent according to the invention and the binding partner or binding partners of the analyte are preferably packaged in separate containers. If an interference-eliminating agent according to the invention is used in which the biotin binding site has been inactivated, the interference-eliminating agent can also be added directly to the binding partners of the analyte.
The invention is elucidated by the following examples.
Example 1 Production of polymeric streptavidin Polymerized streptavidin is produced according to EP-A-0 331 127.
Activation of streptavidin with maleimido-hexanoyl-N-hydroxysuccinimide ester 30 mg streptavidin is dissolved in 3 ml 30 mM potassium phosphate/100 mM sodium chloride (pH 7.1) and heated to 25°C. 0.15 ml maleinimido-hexanoyl-N-hydroxysuccinimide ester (MHS) (Boehringer Mannheim GmbFI) in DMSO (10 mg/ml) is added dropwise while stirring. After a reaction time of 1 hour at 25°C the solution is cooled in an ice bath. Subsequently the MHS-streptavidin which is formed is twice dialysed at 4°C against 1 liter 50 mM
potassium phosphate/100 mM sodium chloride (pH 5.0).
Activation of streptavidin with 8-acetylmercaptosuccinic acid anhydride 30 mg streptavidin is dissolved in 3 ml 100 mM potassium phosphate (pH 7.8) and heated to 25°C. 0.175 ml S-acetylmercaptosuccinic acid anhydride (SAMBA) in DMSO
(10 mg/ml) is added dropwise while stirring. After a reaction time of 3 hours at 25°C the SAMBA-streptavidin which is formed is twice dialysed at 4°C against 1 liter 50 mM potassium phosphate/2 mM EDTA (pH 6.5).
~
218438b Homogeneous cross-linkage of streptavidin 3 ml of a solution of activated SAMBA-streptavidin (10 mg/ml) is heated to 25°C and admixed with 50 ~tl 1 M
hydroxylamine (pH 6.5). After 30 minutes at 25°C it is diluted by addition of 15 ml 50 mM potassium phosphate/100 mM sodium chloride/1 mM EDTA (pH 6.5). The homogeneous cross-linkage of streptavidin is started by addition of 3 m1 activated MHS-streptavidin (10 mg/ml).
After a reaction time of 2 hours at 25°C while carefully stirring, the reaction is terminated by adding 0.2 ml 100 mM cysteine/HC1. After an incubation time of 30 minutes at 25-°C the pH value of the solution is adjusted to 7.5 by addition of 1 M dipotassium hydrogen phosphate. After addition of 0.2 ml 500 mM iodacetamide it is incubated for a further hour at 25°C. Afterwards it is dialysed twice at 4°C against 3 litres 50 mM
potassium phosphate/100 mM sodium chloride (pH 7.5). The conjugate is concentrated in an ultrafiltration cell after dialysis and lyophilized after addition of sucrose (8 Example 2 Production of thermo-BSA-SA
Thermo-BSA streptavidin (thermo-BSA-SA) is produced according to EP-A-0 331 127.
Cross-linking BSA to thermo-BSA
1.0 g BSA is dissolved in 100 ml 20 mM potassium phosphate buffer pH 7.0 and kept at a temperature of - 18 _ 70°C for 5 hours. Subsequently it is cooled to 20°C and dialysed against 100 mM potassium phosphate buffer pH
often used to reduce interference in immunoassays. This cross-linking leads to so-called heterogeneously cross-linked SA. In this process SA and/or the protein can in turn be polymerized in this complex. Methods for coupling SA to these molecules are known to a person -killed in the art. A coupling with DSS, SAMBA, SATP, MAS, SATA has proven to be particularly suitable. A
complex of BSA and SA or poly SA is particularly suitable as an interference-eliminating agent. Even better effects are achieved when polymerized BSA is coupled to SA or poly SA. The polymerization or cross-linking of BSA can be achieved with the aforementioned methods for cross-linking SA. The most suitable is a polymerization by heat treatment as described in EP-A
0331 127.
Thirdly the modification of SA can be an inactivation of the active centres i.e. the binding sites forbiotin.
This results in the formation of so-called inactivated SA. The inactivation can be achieved by a simple saturation of the binding sites with biotin or with a biotin derivative which is bound by avidin or streptavidin. Due to the very high affinity of SA for biotin hardly any biotin which could eventually lead to interferences is released when used in the immunoassay.
The active centre is preferably covalently modified. In this case one or several amino acid residues in the active centre of SA can be derivatized so that binding to biotin is greatly reduced or completely prevented.
Methods for inactivating the biotin binding site in SA
are known from Gitlin et al., Biochem. J. 269 (1990) _$_ 527-530. Tyrosine residues in the active centre of SA
are preferably modified with p-nitrobenzenesulfonyl fluoride. A further method for inactivating the biotin binding site in SA is to covalently couple biotin to the binding site. Methods for coupling biotin to the active centre of SA are known to a person skilled in the art. A
new method for coupling biotin to SA by means of a new photoactivatable biotin derivative has turned out to be particularly preferable. Photoactivatable biotin derivatives are known. In EP-A-0 155 854 and EP-A-0 187 323 azide-substituted phenyls/nitrophenyls are described which are coupled by an amine-containing linker to biotin. Biotin-DAD00-AB (biotin-[8-(4-azidobenzoyl)-amino-3,6-dioxaoctyl]amide) is preferably used as the biotin derivative. Further biotin derivatives are described in the Boehringer Mannheim Biochemica catalogue order no. 1292633 and 1292641. After saturation of the biotin binding sites of SA with the biotin derivatives, the photoreaction is initiated and thus biotin is covalently fixed to the active centre. In this inactivated SA biotin is bound to the active centre and additionally bound outside the active centre via a covalent bond. The SA biotin product obtainable by this means is also a subject matter of the present invention.
A further method of inactivating the active centre of SA
is to modify the binding sites by genetic engineering.
The active centre of SA can be modified and inactivated by substitution, deletion or insertion of individual amino acid residues or short sections of amino acid residues. Preferably individual amino acids such as tyrosine residues are substituted by other amino acids in the active centre. It is, however, also possible to produce SA in which the binding site is partially or completely absent. Methods for modifying and producing SA by means of genetic engineering are known to a person skilled in the art. For example the production of recombinant streptavidin is described in EP-A-0 198 015.
Fourthly the modification can be achieved by a fragmentation of SA. The SA fragments can be produced by chemical or enzymatic cleavage or by recombinant production. In-this case the biotin binding site in particular may be absent as already described above. The advantage of fragmentation is an improved solubility of the product. Preferably all fragments obtained by chemical or enzymatic cleavage from avidin or streptavidin are used in the mixture so that all or nearly all parts of SA are present.
The SA inactivated or fragmented in this way can be used in the aforementioned first or second method for modifying SA i.e. the inactivated SA can in turn be polymerized or cross-linked with other molecules such as BSA or poly BSA. These interference-eliminating agents in which a combination of at least two of the said methods have been carried out to modify the SA have proven to be particularly advantageous.
Afterthe modification of SA by one of the said methods has been completed it is possible to remove remaining biotin binding activity, biotin covalently bound to the surface which is not bound in the active centre of -avidin or streptavidin or free biotin by a suitable purification of the avidin or streptavidin derivatives.
This can for example be achieved by adsorption to solid phase bound biotin and/or SA.
The invention therefore in addition concerns avidin or - to - 218~38~
streptavidin derivatives obtainable by one of the methods described above for modification and subsequent purification to remove remaining biotin binding activity, biotin covalently bound to the surface which is not bound in the active centre of avidin or streptavidin or free biotin preferably by adsorption to a solid phase bound biotin and/or SA.
Furthermore the invention concerns a process for the production of the interference-eliminating agents -according to the invention in which SA is modified according to at least one of the three aforementioned methods and is optionally subsequently purified as described above to remove remaining biotin binding capacity, biotin bound covalently to the surface or free biotin.
Avidin or streptavidin or derivatives thereof can be used to reduce interference in all common immunoassays or nucleic acid assays. They are particularly suitable for reducing interference in immunoassays or nucleic acid assays in which avidin or streptavidin is used as a binding component. Such immunoassays are for example known from Guesdon et al., J. Histochem. Cytochem. 27 (1979) 1131-1139 and Bayer and Wilchek, Analytical Biochemistry 171 (1988) 1-32. The interferences can for example be caused by antibodies against avidin or streptavidin which sometimes occur in human serum.
However, the interference-eliminating agent according to the invention also exhibits an advantageous effect in immunoassays in which no avidin or streptavidin is used.
In these immunoassays the use of SA which is bound to a further molecule such as for example BSA or poly BSA
corresponding to the second method of modification described above has proven to be particularly advantageous in these immunoassays. The interference- _ eliminating agent according to the invention is always used in addition to the reagents that are otherwise necessary in the test. It is not identical to the avidin or streptavidin components which may possibly be present in the test such as an SA-coated solid phase or enzyme-SA conjugates. In contrast to these SA reagents the interference-eliminating reagent according to the invention is not incorporated into the complex to be detected composed of analyte and specific binding partners.
The invention furthermore concerns a method for the determination of an analyte in a sample by (1) contacting the sample with (a) avidin or streptavidin or a derivative thereof (b) one or several specific binding partners of the analyte and (2) measuring the complex formed from analyte and specific binding partners as a measure for the presence of the analyte.
All substances can serve as the analyte which react to form a complex with at least one specific binding partner such as for example haptens, antigens, antibodies or nucleic acids. The method according to the invention is particularly suitable for the detection of antibodies in particular autoantibodies.
In general body fluids such as blood, plasma, serum, -1z-saliva or urine serve as the sample.
Any biological or chemical binding partners can serve as the specific binding partner which is capable of specifically binding the analyte and can form a complex with it. These include antibodies, antibody fragments, antigens, haptens, hormones, avidin, biotin, nucleic acids, oligonucleotides or derivatives thereof.
Antibodies or antigens or fragments thereof are preferably used in the present invention as a binding partner of the analyte.
In order to detect the complex composed of analyte and specific binding partner it is possible to use all methods familiar to a person skilled in the art. It is possible to use homogeneous methods in which all binding partners in the method are in a soluble form such as precipitation methods with a turbidimetric or nephelometric determination of the complex formed or immunoassays based on the CEDIA, EMIT or FPIA principle.
Heterogeneous methods are also suitable in which at least one reagent is bound to a solid phase. Examples of this are agglutination tests in which one partner of a binding pair is for example bound to latex, sandwich assays, ELISA for the detection of specific antibodies such as e.g. against HIV, HCV, rubella, toxoplasma gondii, glutamate decarboxylase or thyroglobulin, RIA or immunometric assays. Apart from the precipitation methods, one of the specific binding partners is labelled in all these methods. The label can directly generate a measurable signal which is for example a radioisotope, a chemiluminescent, fluorescent or electrochemiluminescent label or a coloured particle such as a metal sol particle or dyed or undyed latex.
The label can also generate an indirect signal such as an enzyme label like peroxidase, glucose oxidase, f3-galactosidase or alkaline phosphatase.
The immunoassays can also be carried out by means of test strips or biosensors in particular when one of the binding partners is coupled via SA to the solid phase.
Interference in immunoassays based on the principle plasmon resonance can also be reduced according to the invention.
One reagent of the method for detecting the analyte is often coupled via a specific binding pair such avidin or streptavidin/biotin to the solid-phase. The advantage of this is that the solid phase can be used universally in several test procedures. It is also possible to bind the ._.
label to a component of the assay via a specific binding pair. For example an enzyme can be coupled to avidin or streptavidin and the binding partner, for example an antibody, can be biotinylated. Examples of such test procedures are known to a person skilled in the art. The avidin or streptavidin derivative according to the invention is particularly suitable for reducing interference in those methods of detection which utilize an indirect binding of a reaction component by avidin/
biotin or streptavidin/biotin.
The method for detecting the analyte can be carried out in one or several steps i.e. the incubation of the analyte with the individual test components can be carried out simultaneously or successively. If avidin or streptavidin or a derivative thereof is used in which the biotin binding sites have not been inactivated, then in methods in which a binding component is coupled via avidin/biotin or streptavidin/biotin care must be taken i that binding of the binding components via avidin/biotin or streptavidin/biotin is already completed before avidin or streptavidin or a derivative thereof is added since otherwise the non-inactivated biotin binding sites would bind to the biotinylated binding component and cause interferences. For example when using an avidin or streptavidin solid phase, the biotinylated specific binding partner of the analyte must be added first before the sample is added together with avidin or streptavidin or a derivative thereof. If an avidin or streptavidin derivative according to the invention is used in which the biotin binding sites have been inactivated then all test reagents can be incubated simultaneously since the biotinylated reagents do not bind to the avidin or streptavidin derivative.- This inactivated avidin or streptavidin derivative can thus be used universally in all conceivable test variants and is thus preferred.
The concentration of the interference-eliminating agent according to the invention in the test mixture is -between 0.0001 and 1 ~ (m/v) preferably between 0.01 and 1 ~ (m/v).
The individual reaction components of the method for detecting the analyte are advantageously offered in the form of a test combination or a test kit. Therefore a further subject matter of the invention is a test combination for a method for detecting an analyte in a sample containing avidin or streptavidin or a derivative thereof and at least one specific binding partner of the analyte. In addition it can also contain all other reagents necessary to carry out the test procedure such as buffers, detergents, labels, auxiliary substances to detect the label such as enzyme substrates, solid phases 218438b etc.. The interference-eliminating agent according to the invention and the binding partner or binding partners of the analyte are preferably packaged in separate containers. If an interference-eliminating agent according to the invention is used in which the biotin binding site has been inactivated, the interference-eliminating agent can also be added directly to the binding partners of the analyte.
The invention is elucidated by the following examples.
Example 1 Production of polymeric streptavidin Polymerized streptavidin is produced according to EP-A-0 331 127.
Activation of streptavidin with maleimido-hexanoyl-N-hydroxysuccinimide ester 30 mg streptavidin is dissolved in 3 ml 30 mM potassium phosphate/100 mM sodium chloride (pH 7.1) and heated to 25°C. 0.15 ml maleinimido-hexanoyl-N-hydroxysuccinimide ester (MHS) (Boehringer Mannheim GmbFI) in DMSO (10 mg/ml) is added dropwise while stirring. After a reaction time of 1 hour at 25°C the solution is cooled in an ice bath. Subsequently the MHS-streptavidin which is formed is twice dialysed at 4°C against 1 liter 50 mM
potassium phosphate/100 mM sodium chloride (pH 5.0).
Activation of streptavidin with 8-acetylmercaptosuccinic acid anhydride 30 mg streptavidin is dissolved in 3 ml 100 mM potassium phosphate (pH 7.8) and heated to 25°C. 0.175 ml S-acetylmercaptosuccinic acid anhydride (SAMBA) in DMSO
(10 mg/ml) is added dropwise while stirring. After a reaction time of 3 hours at 25°C the SAMBA-streptavidin which is formed is twice dialysed at 4°C against 1 liter 50 mM potassium phosphate/2 mM EDTA (pH 6.5).
~
218438b Homogeneous cross-linkage of streptavidin 3 ml of a solution of activated SAMBA-streptavidin (10 mg/ml) is heated to 25°C and admixed with 50 ~tl 1 M
hydroxylamine (pH 6.5). After 30 minutes at 25°C it is diluted by addition of 15 ml 50 mM potassium phosphate/100 mM sodium chloride/1 mM EDTA (pH 6.5). The homogeneous cross-linkage of streptavidin is started by addition of 3 m1 activated MHS-streptavidin (10 mg/ml).
After a reaction time of 2 hours at 25°C while carefully stirring, the reaction is terminated by adding 0.2 ml 100 mM cysteine/HC1. After an incubation time of 30 minutes at 25-°C the pH value of the solution is adjusted to 7.5 by addition of 1 M dipotassium hydrogen phosphate. After addition of 0.2 ml 500 mM iodacetamide it is incubated for a further hour at 25°C. Afterwards it is dialysed twice at 4°C against 3 litres 50 mM
potassium phosphate/100 mM sodium chloride (pH 7.5). The conjugate is concentrated in an ultrafiltration cell after dialysis and lyophilized after addition of sucrose (8 Example 2 Production of thermo-BSA-SA
Thermo-BSA streptavidin (thermo-BSA-SA) is produced according to EP-A-0 331 127.
Cross-linking BSA to thermo-BSA
1.0 g BSA is dissolved in 100 ml 20 mM potassium phosphate buffer pH 7.0 and kept at a temperature of - 18 _ 70°C for 5 hours. Subsequently it is cooled to 20°C and dialysed against 100 mM potassium phosphate buffer pH
7.8.
Activation of thermo-BSA with SAMBA
68 mg thermo-BSA is dissolved in 2 ml 0.1 M potassium phosphate buffer pH 7.8 and it is slowly admixed with 0.38 ml SAMBA (10 mg/ml in DMSO). After a reaction time of 3.5 hours at 25°C it is dialysed at 4°C against 1 liter.50 mM potassium phosphate buffer pH 6.5.
Production of a thermo-BSA-streptavidin conjugate The heterogeneous cross-linking of streptavidin with thermo-BSA is carried out analogously to the homogeneous cross-linking described in example 1. In this process-60 mg activated MHS-streptavidin (produced according to -example 1) is reacted with 68 mg SAMBA-thermo-BSA. The reaction product is purified by means of gel filtration (Superose 6 prep. grade) and concentrated in an ultrafiltration cell. The product obtained is subsequently lyophilized.
Example 3 saturation of thermo-BSA-8A with free biotin 60 mg thermo-BSA-SA dissolved in 6.0 ml potassium phosphate buffer, 100 mM pFi 7.0 is admixed with 2 mg D-biotin dissolved in 0.5 ml potassium phosphate buffer, mM pH 7.8 and stirred for 1 hour.
218438b The free biotin is separated by means of gel filtration (Superose G~). The high molecular protein fraction is dialysed against 10 mM potassium phosphate buffer, pfi 7.0 and lyophilized after addition of 8 °s sucrose. _ Example 4 Production of covalently-modified streptavidin Tyrosine residues in the active centre of streptavidin/avidin are derivatized with p-nitrobenzene-sulfonyl fluoride according to G. Gitlin, E.A. Bayer, M.
Wilchek: Studies on the biotin-binding sites of Avidin and Streptavidin; Biochem. J. (1990), 2G9, 527-530.
A 200-fold molar excess of p-nitro-benzenesulfonyl fluoride (Sigma N-2262) relative to the streptavidin subunit is added to 1 g streptavidin at a protein concentration of l0 mg/ml in 0.1 M Tris-iiCl buffer, pH
7.9. After the addition it is stirred for a further 18 -20 hours at 25°C. The product is then dialysed against > 500-fold volume PBS buffer pH 7.5 (16 - 18 hours at 4°) in order to separate non-reacted derivatization reagent.
Example 5 Production of photoaotivatable biotin Synthesis of biotin-[8-(4-azidobenzoyl)amino-3,6-dioxaoctyl]amide (biotin-DADOO-AB) 1.50 g (4 mmol) biotinoyl-1,8-diamino-3,6-dioxaoctane (biotin-DADOO, Boehringer Mannheim GmbH) is dissolved in 50 ml freshly distilled DMF while stirring. 1.04 g (4 mmol) N-hydroxysuccinimidyl-(4-azidobenzoate) (HSAB, Boehringer Mannheim GmbH) and 0.55 ml (4 mmol) triethylamine is successively added to the solution and allowed to stir for 2 hours at 20°C. Subsequently the solvent is removed on a rotary evaporator in an oil pump vacuum and the crude product that remains is purified by chromatography on silica gel. For this it is dissolved in as small amount as possible of chloroform/methanol 2/1 (v/v) while heating slightly to ca. 40°C and applied to a silica gel 60 (Merck Company, Germany) column (4 x 60 cm). It is eluted with chloroform/methanol 2/1 (v/v) and fractions of 50 ml are pooled. The fractions containing the pure product are determined by means of TLC (system as described below) and pooled. The solvent is removed on a rotary evaporator and the semi-solid residue is digested with ca. 50 ml diisopropyl ether.
The finely crystalline, colourless product is suction filtered and dried overnight in a vacuum drying oven (0.1 - 0.15 bar/40°C).
Yield: 1.24 g (60 ~ of theory) TLC: silica gel 60 (Merck)F254, chloroform/
methanol 2/1 (v/v);
Rg = 0.71.
218438b 1H-NMR(100MHz/d6-DMS0:8 (ppm) = 1.20-1.65 (m,6H); 2.07 (tr,2H); 2.60-3.65 (m,l5H); 4.05-4.20 (m;2H); 6.38 (d,br, 2H), 7.20 (d, 2H), 7.62 (tr,br,iH); 7.91 (d, 2H); 8.53 (tr,br,iH).
UV (CH30H): ~(max) = 267nm IR(KBr) : a = 2125- c~a 1 The synthetic path of biotin-DADOO-AB is shown in Figure 1.
Example 6 Production of biotin(photoactivated) streptavidin Streptavidin is reacted with a photoactivatable biotin derivative (e. g. biotin-DAD00-AB) and dialysed to remove free unbound biotin. The photoreaction is initiated by irradiation with a Hg vapour lamp (350-700 nm) and the biotin is covalently immobilized in the binding centre of streptavidin.
A 10-fold molar excess of biotin-DAD00-AB reagent (3.5 ml of a 25 mg/ml biotin-DADOO-AB stock solution in DMSO) is added to 1 g streptavidin at a protein concentration of 20 mg/ml in PBS buffer pH 7.5. After ___ the addition it is stirred for 2 hours at 25°C with exclusion of light.
Free. unbound biotin derivative is completely separated (no longer detectable) by dialysis (20 hours, 4°C) _ 22 against a > 500-fold volume of PBS buffer, pft 7.5 with exclusion of light. The mixture is then irradiated for 20 min with a c 5 cm path length of the solution using a Hg vapour lamp (350-700 nm) while stirring and subsequently dialysed again against a > 500-fold volume of PBS buffer, pFt 7.5 (16-18 hours, ~°C).
Example 7 Purification of inactivated streptavidin and thermo-BSA-sA and derivatives and fragments thereof Production of BSA-biotin adsorber A 10-fold molar excess of D-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester (Boehringer Mannheim GmbH) was added to 1 g BSA at a protein concentration of l0 mg/ml in PBS buffer, pfi 8.5.
After the addition it is stirred for 2 hours at 25°C and the reaction is stopped by addition of lysine to a final concentration of 10 mM. Free unbound biotin derivative is completely separated (no longer detectable) by dialysis (16-18 h, 4°C) against a > 500-fold-volume of PBS buffer, pfI 7.5.
300 ml glutaric dialdehyde (10 a) is added to 40 g amino-Spherosil~ (Boehringer Mannheim Gmbtl) and the mixture is stirred for 2 hours at pH 3.7 and 55°C while -.
rotating. The suspension is washed with > 7 Spherosil~-volumes of redistilled water and 5 Spherosil~ volumes of PBS buffer, pH 8Ø The activated Spherosil" is subsequently reacted with BSA-Bi for 20 hours at room temperature with a protein supply of 5 - 10 mg/ml Spherosil while shaking.
The unreacted protein solution is separated over a glass suction filter and the adsorber material is washed with Spherosil~ volumes 0.9 % kdaCl solution and incubated for 1 hour with 5 Spherosil~ volumes of ethanolamine solution.
The adsorber material is then washed with a 5-fold Spherosil~ volume of 0.9 % IIaCl solution, washed with a 3-fold Spherosil" volume of 1M propionic acid and enough 30 mM NaCl solution to reach a pii of 6.5. The adsorber is adequately equilibrated with PBS buffer pH 7.5.
Production of streptavidin-8pherosil adsorber 300 m1 glutaric dialdehyde (10 %) is added to 40 g amino-Spherosil (Boehringer Mannheim Gmbil) and the mixture is stirred for 2 hours at pll 3.7 and 55°C while rotating. The suspension is washed with > 7 Spherosil volumes of redistilled water and 5 Spherosil volumes of PBS buffer, pFi 5Ø The activated Spherosil is _ subsequently reacted with streptavidin for 20 hours at room temperature with a protein supply of 5 - 10 mg/ml Spherosil (Boehringer Mannheim GmbH) while shaking.
The unreacted protein solution is separated over a glass suction filter and the adsorber material is washed with 10 Spherosil volumes of 0.9 % NaCl solution and incubated for 1 hour with 5 volumes ethanolamine solution. The adsorber material is then washed with the 5-fold Spherosil volume of 0.9 % NaCI solution, with the 3-fold Spherosil volume of 1M propionic acid and enough 30 mM NaCl solution to reach a pIF of 6.5. The adsorber is adequately equilibrated with PBS buffer pFI 7.5.
Inactivated streptavidin is purified-of streptavidin with residual activity (biotin binding), residual free biotin or streptavidin having biotin that is covalently accessible on the surface (in contrast to biotin immobilized in the binding pocket) according to example 3, 4 or 6 by means of chromatography on a bovine serum albumin-biotin and/or streptavidin adsorber based on Spherosil. -1 ml streptavidin-Spherosil adsorber, equilibrated in PBS buffer pH 7.5, is added to the reaction mixture per mg protein and stirred for 2 hours at 25°C.
The suspension is then transferred to a column and the column material is washed with PBS buffer pH 7.5. In this process the protein content is monitored at the outlet of the column via a UV monitor at AZgonm~ It is washed until free of protein. The eluant containing protein is collected in a fraction.
1 ml bovine serum albumin-biotin (BSA-Bi)-Spherosil adsorber, equilibrated in PBS buffer, pH 7.5 is added per 10 mg protein to the eluant of the streptavidin adsorber containing protein and it is stirred for 2 hours at room temperature.
The suspension is transferred to a column and washed with PBS buffer, pH 7.5. In this process the protein content is monitored at the outlet of the column via a UV monitor at A2aonm~ The eluant containing protein contains the product and is collected as a fraction. The product (inactivated (poly)streptavidin or thermo-BSA-SA
or derivatives and fragments thereof] is concentrated to _ a protein concentration of 20 mg/ml and lyophilized after dispensing.
Example 8 Carrying out a GAD antibody test in a sequential test procedure with regard to biotinylated antigen and sample 100 ~.1 biotinylated glutamate decarboxylase (GAD) isolated from pig brain is pipetted at an optimal concentration for each of the respective batches (1-3 ~,g/ml in the incubation buffer from the Boehringer Mannheim Enzymun Test~ Anti-HIV 1+2) into each well of a microtitre plate precoated with thermo-BSA
streptavidin and incubated for 30 min at room temperature. Afterwards the plate is washed three times with 350 ~C1 50 mmol/1 potassium phosphate, pH 7.0 with addition of 0.1 % (w/v) CHAPSO (3-(3-cholamidopropyl)-dimethylammonioJ-2-hydroxy-1-propanesulfonate) each time. Human serum is diluted 1+25 in incubation buffer from the Enzymun Test~ anti-HIV 1+2 to which the respective amount of-thermo-BSA-streptavidin (untreated or inactivated) or streptavidin monomer or streptavidin polymer (untreated or inactivated) was added. These samples diluted in this way are incubated for 1 hour at room temperature in the microtitre plate in a volume of 100 ~1/well while shaking. Subsequently the samples are aspirated and the plates are washed three times as above. The POD-coupled detection antibody from sheep with a binding specificity for human IgG is diluted in conjugate buffer from the Enzymun Test~ anti-HIV 1+2 to a concentration of 75 mU/ml and 100 ~C1 of this diluted solution is incubated in each well for 1 hour at room temperature-while shaking at room temperature. The liquid is aspirated and the plate is again washed 3 times as above. The dye ABTS~ is dissolved to a concentration of 1 mg/ml in the Enzymun Test~ substrate buffer and 100 Wl/well is incubated at room temperature without shaking. After ca. 30 min the absorbance is read in a microtitre plate photometer. The measuring wavelength is 405 nm and the reference wavelength is 492 nm. The blank is two untreated wells which only contain substrate/dye solution. The mean value of the absorbance of the two blank wells is deducted from all other absorbances.
The mean values of a sample determined in duplicate from two wells are listed as absorbances in tables 2, 3 and 4.
- 2' - 2184386 Table 2 Interference elimination by addition of various amounts of thermo-B8A streptavidin Sample AbsorbanceSignal level in %
of the initial signal with without the respective amount of thermo-BSA-SA
(%
w/v) addition to buffer 0 0.01 0.025 0.05 0.1 0.25 0.5 1 buffer 0.016 normal serum 0.182 100 95 68 71 74 70 70 60 positive 1.906 100 72 74 73.5 73 65 62 44 control (MICA) positive serum2.065 100 63 61 62 60 50 48 37 interference 2.325 100 16 14 15 16 15 14 11 serum 1 interference 1.243 100 9 8.5 8 5 4 4 2 serum 2 interference 0.707 100 24 16 13 13 10 10 8 serum 3 * not listed because inappropriate due to the low measured signal.
MICA: monoclonal antibody against GAD
Table 3 Interference elimination by addition of monomeric or polymeric streptavidin sample without 1 %(w/v)SA 1%(w/v) polySA
addition buffer 0.008 0.006 0.009 normal serum 0.106 0.144 0.108 interference 1.390 0.108 0.087 serum positive 0.950 1.588 1.033 control (MICA) positive serum 0.940 1.416 0.950 Table 4 Interference elimination by addition of thermo-BSA-streptavidin or thermo-BSA-streptavidin which had been covalently modified (according to example 4) Sample without 0.025 %(w/v) 0.025 %(w/v) addition thermo-BSA-SA thermo-BSA-SA
(inact.) buffer 0.009 0.011 0.011 normal serum 0.226 0.191 0.206 interference 1.500 0.117 0.332 serum positive 1.745 1.528 1.579 control (MICA) positive serum 1.963 1.457 0.797 Example 9 Elimination of interference in an anti-HCV test using biotin(photoactivated)-SA
Test principle:
2-Step sandwich assay with a streptavidin solid phase (test procedure and reagents as in the-Boehringer Mannheim Enzymun Test~ anti-HIV 1+2) 1st Step: biotinylated peptidesplus sample 2nd Step: reaction of wall-bound antibody with an anti-human IgG-POD conjugate 3rd Step: indicator reaction with ABTS as the substrate Buffer:
a) Incubation buffer from the Enzymun Testy anti-HIV
1+2 HCV peptides from the core, NS4 and NS5 region ~ inactivated streptavidin prepared according to example 6 and 7 b) Conjugate buffer from the Enzymun Test~ anti-HIV
1+2 w Incubation periods:
1st Step: 1 hour (sample + incubation buffer) 2nd Step: 1 hour (+ conjugate buffer) 3rd Step: 1 hour (substrate reaction with ABTS) Samples:
3 negative serum samples (reference 1) 6 false-positive anti-HCV negative samples 3 positive anti-HCV samples (reference 2) Volumes:
Sample 20 ~1 all other reagents 500 ~1 of each Test procedure: __ On an ES 600 at 25°C according to the test instructions of the Enzymun Test~ anti-HIV 1+2 Substrate measurement:, Measurement of the substrate solution at 422 nm on an ES
600 (Boehringer Mannheim GmbH). The absorbances are listed in table 5.
Table 5 Samples without 20 ~CgJml decrease in inact. SA in inact. SA in signal after incubation incubation addition of buffer buffer inact. SA
negative serum 0.036 0.027 l negative serum 0.042 0.035 negative serum 0.078 0.076 *
HCV negative 0.528 0.125 serum 1 HCV negative 0.467 0.106 77 0 serum 2 HCV negative . 0.979 0.161 84 serum 3 HCV negative 0.499 0.094 81 serum 4 HCV negative 2.049 0.471 serum 5 HCV negative 0.427 0065 85 's serum 6 HCV positive 1.747 1.645 6 ~
serum 1 HCV positive 1.161 1.076 serum 2 HCV positive 1.104 1.026 serum 3 * not stated, inappropriate due to the low measured signal.
Activation of thermo-BSA with SAMBA
68 mg thermo-BSA is dissolved in 2 ml 0.1 M potassium phosphate buffer pH 7.8 and it is slowly admixed with 0.38 ml SAMBA (10 mg/ml in DMSO). After a reaction time of 3.5 hours at 25°C it is dialysed at 4°C against 1 liter.50 mM potassium phosphate buffer pH 6.5.
Production of a thermo-BSA-streptavidin conjugate The heterogeneous cross-linking of streptavidin with thermo-BSA is carried out analogously to the homogeneous cross-linking described in example 1. In this process-60 mg activated MHS-streptavidin (produced according to -example 1) is reacted with 68 mg SAMBA-thermo-BSA. The reaction product is purified by means of gel filtration (Superose 6 prep. grade) and concentrated in an ultrafiltration cell. The product obtained is subsequently lyophilized.
Example 3 saturation of thermo-BSA-8A with free biotin 60 mg thermo-BSA-SA dissolved in 6.0 ml potassium phosphate buffer, 100 mM pFi 7.0 is admixed with 2 mg D-biotin dissolved in 0.5 ml potassium phosphate buffer, mM pH 7.8 and stirred for 1 hour.
218438b The free biotin is separated by means of gel filtration (Superose G~). The high molecular protein fraction is dialysed against 10 mM potassium phosphate buffer, pfi 7.0 and lyophilized after addition of 8 °s sucrose. _ Example 4 Production of covalently-modified streptavidin Tyrosine residues in the active centre of streptavidin/avidin are derivatized with p-nitrobenzene-sulfonyl fluoride according to G. Gitlin, E.A. Bayer, M.
Wilchek: Studies on the biotin-binding sites of Avidin and Streptavidin; Biochem. J. (1990), 2G9, 527-530.
A 200-fold molar excess of p-nitro-benzenesulfonyl fluoride (Sigma N-2262) relative to the streptavidin subunit is added to 1 g streptavidin at a protein concentration of l0 mg/ml in 0.1 M Tris-iiCl buffer, pH
7.9. After the addition it is stirred for a further 18 -20 hours at 25°C. The product is then dialysed against > 500-fold volume PBS buffer pH 7.5 (16 - 18 hours at 4°) in order to separate non-reacted derivatization reagent.
Example 5 Production of photoaotivatable biotin Synthesis of biotin-[8-(4-azidobenzoyl)amino-3,6-dioxaoctyl]amide (biotin-DADOO-AB) 1.50 g (4 mmol) biotinoyl-1,8-diamino-3,6-dioxaoctane (biotin-DADOO, Boehringer Mannheim GmbH) is dissolved in 50 ml freshly distilled DMF while stirring. 1.04 g (4 mmol) N-hydroxysuccinimidyl-(4-azidobenzoate) (HSAB, Boehringer Mannheim GmbH) and 0.55 ml (4 mmol) triethylamine is successively added to the solution and allowed to stir for 2 hours at 20°C. Subsequently the solvent is removed on a rotary evaporator in an oil pump vacuum and the crude product that remains is purified by chromatography on silica gel. For this it is dissolved in as small amount as possible of chloroform/methanol 2/1 (v/v) while heating slightly to ca. 40°C and applied to a silica gel 60 (Merck Company, Germany) column (4 x 60 cm). It is eluted with chloroform/methanol 2/1 (v/v) and fractions of 50 ml are pooled. The fractions containing the pure product are determined by means of TLC (system as described below) and pooled. The solvent is removed on a rotary evaporator and the semi-solid residue is digested with ca. 50 ml diisopropyl ether.
The finely crystalline, colourless product is suction filtered and dried overnight in a vacuum drying oven (0.1 - 0.15 bar/40°C).
Yield: 1.24 g (60 ~ of theory) TLC: silica gel 60 (Merck)F254, chloroform/
methanol 2/1 (v/v);
Rg = 0.71.
218438b 1H-NMR(100MHz/d6-DMS0:8 (ppm) = 1.20-1.65 (m,6H); 2.07 (tr,2H); 2.60-3.65 (m,l5H); 4.05-4.20 (m;2H); 6.38 (d,br, 2H), 7.20 (d, 2H), 7.62 (tr,br,iH); 7.91 (d, 2H); 8.53 (tr,br,iH).
UV (CH30H): ~(max) = 267nm IR(KBr) : a = 2125- c~a 1 The synthetic path of biotin-DADOO-AB is shown in Figure 1.
Example 6 Production of biotin(photoactivated) streptavidin Streptavidin is reacted with a photoactivatable biotin derivative (e. g. biotin-DAD00-AB) and dialysed to remove free unbound biotin. The photoreaction is initiated by irradiation with a Hg vapour lamp (350-700 nm) and the biotin is covalently immobilized in the binding centre of streptavidin.
A 10-fold molar excess of biotin-DAD00-AB reagent (3.5 ml of a 25 mg/ml biotin-DADOO-AB stock solution in DMSO) is added to 1 g streptavidin at a protein concentration of 20 mg/ml in PBS buffer pH 7.5. After ___ the addition it is stirred for 2 hours at 25°C with exclusion of light.
Free. unbound biotin derivative is completely separated (no longer detectable) by dialysis (20 hours, 4°C) _ 22 against a > 500-fold volume of PBS buffer, pft 7.5 with exclusion of light. The mixture is then irradiated for 20 min with a c 5 cm path length of the solution using a Hg vapour lamp (350-700 nm) while stirring and subsequently dialysed again against a > 500-fold volume of PBS buffer, pFt 7.5 (16-18 hours, ~°C).
Example 7 Purification of inactivated streptavidin and thermo-BSA-sA and derivatives and fragments thereof Production of BSA-biotin adsorber A 10-fold molar excess of D-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester (Boehringer Mannheim GmbH) was added to 1 g BSA at a protein concentration of l0 mg/ml in PBS buffer, pfi 8.5.
After the addition it is stirred for 2 hours at 25°C and the reaction is stopped by addition of lysine to a final concentration of 10 mM. Free unbound biotin derivative is completely separated (no longer detectable) by dialysis (16-18 h, 4°C) against a > 500-fold-volume of PBS buffer, pfI 7.5.
300 ml glutaric dialdehyde (10 a) is added to 40 g amino-Spherosil~ (Boehringer Mannheim Gmbtl) and the mixture is stirred for 2 hours at pH 3.7 and 55°C while -.
rotating. The suspension is washed with > 7 Spherosil~-volumes of redistilled water and 5 Spherosil~ volumes of PBS buffer, pH 8Ø The activated Spherosil" is subsequently reacted with BSA-Bi for 20 hours at room temperature with a protein supply of 5 - 10 mg/ml Spherosil while shaking.
The unreacted protein solution is separated over a glass suction filter and the adsorber material is washed with Spherosil~ volumes 0.9 % kdaCl solution and incubated for 1 hour with 5 Spherosil~ volumes of ethanolamine solution.
The adsorber material is then washed with a 5-fold Spherosil~ volume of 0.9 % IIaCl solution, washed with a 3-fold Spherosil" volume of 1M propionic acid and enough 30 mM NaCl solution to reach a pii of 6.5. The adsorber is adequately equilibrated with PBS buffer pH 7.5.
Production of streptavidin-8pherosil adsorber 300 m1 glutaric dialdehyde (10 %) is added to 40 g amino-Spherosil (Boehringer Mannheim Gmbil) and the mixture is stirred for 2 hours at pll 3.7 and 55°C while rotating. The suspension is washed with > 7 Spherosil volumes of redistilled water and 5 Spherosil volumes of PBS buffer, pFi 5Ø The activated Spherosil is _ subsequently reacted with streptavidin for 20 hours at room temperature with a protein supply of 5 - 10 mg/ml Spherosil (Boehringer Mannheim GmbH) while shaking.
The unreacted protein solution is separated over a glass suction filter and the adsorber material is washed with 10 Spherosil volumes of 0.9 % NaCl solution and incubated for 1 hour with 5 volumes ethanolamine solution. The adsorber material is then washed with the 5-fold Spherosil volume of 0.9 % NaCI solution, with the 3-fold Spherosil volume of 1M propionic acid and enough 30 mM NaCl solution to reach a pIF of 6.5. The adsorber is adequately equilibrated with PBS buffer pFI 7.5.
Inactivated streptavidin is purified-of streptavidin with residual activity (biotin binding), residual free biotin or streptavidin having biotin that is covalently accessible on the surface (in contrast to biotin immobilized in the binding pocket) according to example 3, 4 or 6 by means of chromatography on a bovine serum albumin-biotin and/or streptavidin adsorber based on Spherosil. -1 ml streptavidin-Spherosil adsorber, equilibrated in PBS buffer pH 7.5, is added to the reaction mixture per mg protein and stirred for 2 hours at 25°C.
The suspension is then transferred to a column and the column material is washed with PBS buffer pH 7.5. In this process the protein content is monitored at the outlet of the column via a UV monitor at AZgonm~ It is washed until free of protein. The eluant containing protein is collected in a fraction.
1 ml bovine serum albumin-biotin (BSA-Bi)-Spherosil adsorber, equilibrated in PBS buffer, pH 7.5 is added per 10 mg protein to the eluant of the streptavidin adsorber containing protein and it is stirred for 2 hours at room temperature.
The suspension is transferred to a column and washed with PBS buffer, pH 7.5. In this process the protein content is monitored at the outlet of the column via a UV monitor at A2aonm~ The eluant containing protein contains the product and is collected as a fraction. The product (inactivated (poly)streptavidin or thermo-BSA-SA
or derivatives and fragments thereof] is concentrated to _ a protein concentration of 20 mg/ml and lyophilized after dispensing.
Example 8 Carrying out a GAD antibody test in a sequential test procedure with regard to biotinylated antigen and sample 100 ~.1 biotinylated glutamate decarboxylase (GAD) isolated from pig brain is pipetted at an optimal concentration for each of the respective batches (1-3 ~,g/ml in the incubation buffer from the Boehringer Mannheim Enzymun Test~ Anti-HIV 1+2) into each well of a microtitre plate precoated with thermo-BSA
streptavidin and incubated for 30 min at room temperature. Afterwards the plate is washed three times with 350 ~C1 50 mmol/1 potassium phosphate, pH 7.0 with addition of 0.1 % (w/v) CHAPSO (3-(3-cholamidopropyl)-dimethylammonioJ-2-hydroxy-1-propanesulfonate) each time. Human serum is diluted 1+25 in incubation buffer from the Enzymun Test~ anti-HIV 1+2 to which the respective amount of-thermo-BSA-streptavidin (untreated or inactivated) or streptavidin monomer or streptavidin polymer (untreated or inactivated) was added. These samples diluted in this way are incubated for 1 hour at room temperature in the microtitre plate in a volume of 100 ~1/well while shaking. Subsequently the samples are aspirated and the plates are washed three times as above. The POD-coupled detection antibody from sheep with a binding specificity for human IgG is diluted in conjugate buffer from the Enzymun Test~ anti-HIV 1+2 to a concentration of 75 mU/ml and 100 ~C1 of this diluted solution is incubated in each well for 1 hour at room temperature-while shaking at room temperature. The liquid is aspirated and the plate is again washed 3 times as above. The dye ABTS~ is dissolved to a concentration of 1 mg/ml in the Enzymun Test~ substrate buffer and 100 Wl/well is incubated at room temperature without shaking. After ca. 30 min the absorbance is read in a microtitre plate photometer. The measuring wavelength is 405 nm and the reference wavelength is 492 nm. The blank is two untreated wells which only contain substrate/dye solution. The mean value of the absorbance of the two blank wells is deducted from all other absorbances.
The mean values of a sample determined in duplicate from two wells are listed as absorbances in tables 2, 3 and 4.
- 2' - 2184386 Table 2 Interference elimination by addition of various amounts of thermo-B8A streptavidin Sample AbsorbanceSignal level in %
of the initial signal with without the respective amount of thermo-BSA-SA
(%
w/v) addition to buffer 0 0.01 0.025 0.05 0.1 0.25 0.5 1 buffer 0.016 normal serum 0.182 100 95 68 71 74 70 70 60 positive 1.906 100 72 74 73.5 73 65 62 44 control (MICA) positive serum2.065 100 63 61 62 60 50 48 37 interference 2.325 100 16 14 15 16 15 14 11 serum 1 interference 1.243 100 9 8.5 8 5 4 4 2 serum 2 interference 0.707 100 24 16 13 13 10 10 8 serum 3 * not listed because inappropriate due to the low measured signal.
MICA: monoclonal antibody against GAD
Table 3 Interference elimination by addition of monomeric or polymeric streptavidin sample without 1 %(w/v)SA 1%(w/v) polySA
addition buffer 0.008 0.006 0.009 normal serum 0.106 0.144 0.108 interference 1.390 0.108 0.087 serum positive 0.950 1.588 1.033 control (MICA) positive serum 0.940 1.416 0.950 Table 4 Interference elimination by addition of thermo-BSA-streptavidin or thermo-BSA-streptavidin which had been covalently modified (according to example 4) Sample without 0.025 %(w/v) 0.025 %(w/v) addition thermo-BSA-SA thermo-BSA-SA
(inact.) buffer 0.009 0.011 0.011 normal serum 0.226 0.191 0.206 interference 1.500 0.117 0.332 serum positive 1.745 1.528 1.579 control (MICA) positive serum 1.963 1.457 0.797 Example 9 Elimination of interference in an anti-HCV test using biotin(photoactivated)-SA
Test principle:
2-Step sandwich assay with a streptavidin solid phase (test procedure and reagents as in the-Boehringer Mannheim Enzymun Test~ anti-HIV 1+2) 1st Step: biotinylated peptidesplus sample 2nd Step: reaction of wall-bound antibody with an anti-human IgG-POD conjugate 3rd Step: indicator reaction with ABTS as the substrate Buffer:
a) Incubation buffer from the Enzymun Testy anti-HIV
1+2 HCV peptides from the core, NS4 and NS5 region ~ inactivated streptavidin prepared according to example 6 and 7 b) Conjugate buffer from the Enzymun Test~ anti-HIV
1+2 w Incubation periods:
1st Step: 1 hour (sample + incubation buffer) 2nd Step: 1 hour (+ conjugate buffer) 3rd Step: 1 hour (substrate reaction with ABTS) Samples:
3 negative serum samples (reference 1) 6 false-positive anti-HCV negative samples 3 positive anti-HCV samples (reference 2) Volumes:
Sample 20 ~1 all other reagents 500 ~1 of each Test procedure: __ On an ES 600 at 25°C according to the test instructions of the Enzymun Test~ anti-HIV 1+2 Substrate measurement:, Measurement of the substrate solution at 422 nm on an ES
600 (Boehringer Mannheim GmbH). The absorbances are listed in table 5.
Table 5 Samples without 20 ~CgJml decrease in inact. SA in inact. SA in signal after incubation incubation addition of buffer buffer inact. SA
negative serum 0.036 0.027 l negative serum 0.042 0.035 negative serum 0.078 0.076 *
HCV negative 0.528 0.125 serum 1 HCV negative 0.467 0.106 77 0 serum 2 HCV negative . 0.979 0.161 84 serum 3 HCV negative 0.499 0.094 81 serum 4 HCV negative 2.049 0.471 serum 5 HCV negative 0.427 0065 85 's serum 6 HCV positive 1.747 1.645 6 ~
serum 1 HCV positive 1.161 1.076 serum 2 HCV positive 1.104 1.026 serum 3 * not stated, inappropriate due to the low measured signal.
Claims (26)
1. Use of avidin or streptavidin or a derivative thereof as an interference-eliminating agent to avoid unspecific interactions in immunoassays or nucleic acid assays.
2. Use of avidin or streptavidin or a derivative thereof as claimed in claim 1, wherein homogeneously cross-linked avidin or streptavidin molecules are used as the avidin or streptavidin derivative.
3. Use of avidin or streptavidin or a derivative thereof as claimed in claim 1, wherein avidin or streptavidin molecules cross-linked by bifunctional or polyfunctional compounds are used as the avidin or streptavidin derivative.
4. Use of avidin or streptavidin or a derivative thereof as claimed in claim 1, wherein heterogeneous cross-linked avidin or streptavidin molecules are used as the avidin or streptavidin derivative.
5. Use of avidin or streptavidin or a derivative thereof as claimed in claim 4, wherein avidin or streptavidin molecules cross-linked with proteins are used as the avidin or streptavidin derivative.
6. Use of avidin or streptavidin or a derivative thereof as claimed in claim 5, wherein avidin or streptavidin molecules cross-linked with polymerized proteins are used as the avidin or streptavidin derivative.
7. Use of avidin or streptavidin or a derivative thereof as claimed in claim 5 or 6, wherein homogeneously cross-linked avidin or streptavidin molecules are used as the avidin or streptavidin derivative.
8. Use of avidin or streptavidin or a derivative thereof as claimed in claim 1, wherein fragments or a mixture of fragments of avidin or streptavidin which are able to retain the interference-eliminating effect of the entire molecule are used as the avidin or streptavidin derivative.
9. Use of avidin or streptavidin or a derivative thereof as claimed in claims 1 to 8, wherein inactivated avidin or streptavidin is used as the avidin or streptavidin derivative.
10. Use of avidin or streptavidin or a derivative thereof as claimed in claim 9, wherein the avidin or streptavidin is inactivated by saturation with biotin or a biotin derivative to form said inactivated avidin or streptavidin.
11. Use of avidin or streptavidin or a derivative thereof as claimed in claim 9, wherein the avidin or streptavidin is inactivated by covalently modifying the active centre of avidin or streptavidin to form said inactivated avidin or streptavidin.
12. Use of avidin or streptavidin or a derivative thereof as claimed in claim 11, wherein for the covalent modifi-cation at least one amino acid of the active centre is derivatized or biotin is covalently coupled to the active centre.
13. Use of avidin or streptavidin or a derivative thereof as claimed in claim 12, wherein biotin is covalently coupled to the active centre by photoactivatable biotin.
14. Use of avidin or streptavidin or a derivative thereof as claimed in claim 13, wherein said photoactivatable biotin is biotin-DADOO-AB.
15. Use of avidin or streptavidin or a derivative thereof as claimed in claim 9, wherein the active centre is deactivated by a genetic engineering method comprising substitution, deletion or insertion of individual or several amino acid residues.
16. Use of avidin or streptavidin or a derivative thereof as claimed in any one of claims 9 to 15, wherein the inactivated avidin or streptavidin has been additionally purified over at least one of solid phase bound biotin, avidin and streptavidin.
17. Use of avidin or streptavidin or a derivative thereof as claimed in any one of claims 1 to 16 as an interference-eliminating agent in an immunoassay or a nucleic acid assay in which avidin or streptavidin is used as a binding component.
18. Method for the determination of an analyte in a sample comprising the steps (1) contacting the sample with (a) an interference-eliminating agent as claimed in any one of claims 1 to 16 (b) one or several specific binding partners of the analyte and (2) measuring the complex formed from the analyte and specific binding partner as a measure for the presence of the analyte.
19. Method for the determination of an analyte in a sample as claimed in claim 18, wherein at least one binding component of the method is coupled via avidin/biotin or streptavidin/biotin and the coupling of this binding component is carried out before addition of the interference-eliminating agent.
20. Method for the determination of an analyte in a sample comprising the steps (1) contacting the sample with (a) an interference-eliminating agent as claimed in any one of claims 1 to 16 (b) one or several specific binding partners of the analyte and (2) measuring the complex formed from analyte and specific binding partner as a measure for the presence of the analyte in which the sample is simultaneously contacted with (a) and (b).
21. Test combination for a method for determining an analyte in a sample containing (1) an avidin or streptavidin derivative which is not incorporated into the complex of analyte and analyte binding partner as claimed in any one of claims 1 to 16 and (2) at least one specific binding partner of the analyte.
22. Test combination as claimed in claim 21 additionally containing all further reagents that the method requires for the determination.
23. Process for the production of an interference-eliminating agent as claimed in any one of claims 9 to 16, comprising:
modifying avidin or streptavidin, and purifying the modified avidin or streptavidin over at least one of solid phase-bound biotin, avidin and streptavidin.
modifying avidin or streptavidin, and purifying the modified avidin or streptavidin over at least one of solid phase-bound biotin, avidin and streptavidin.
24. Inactivated avidin or streptavidin obtainable by saturating the active centres with biotin or a biotin derivative or by covalent modification of the active centre and purification over at least one of solid phase bound biotin, avidin and streptavidin.
25. Inactivated avidin or streptavidin in which biotin is bound to the active centre and is additionally bound outside the active centre via a covalent bond obtainable by saturating the active centres of avidin or streptavidin with a photoactivatable biotin derivative and subsequently covalently coupling the biotin derivative by initiating the photoreaction.
26. Inactivated avidin or streptavidin as claimed in claim 25, wherein biotin-DADOO-AB is used as the biotin derivative.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4407423.9 | 1994-03-05 | ||
| DE4407423A DE4407423A1 (en) | 1994-03-05 | 1994-03-05 | Anti-interference agent for use in immunoassays |
| WOPCT/EP95/OO69O | 1995-02-25 | ||
| PCT/EP1995/000690 WO1995023800A1 (en) | 1994-03-05 | 1995-02-25 | Photo-activatable biotin derivatives and their use in suppressing interference in immuno assaying |
| PCT/EP1995/000776 WO1995023801A1 (en) | 1994-03-05 | 1995-03-03 | Interference suppression agent for use in immuno assaying |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2184386A1 CA2184386A1 (en) | 1995-09-08 |
| CA2184386C true CA2184386C (en) | 2005-10-18 |
Family
ID=25934426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002184386A Expired - Lifetime CA2184386C (en) | 1994-03-05 | 1995-03-03 | Interference eliminating agent for application in immunoassays |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US5863740A (en) |
| JP (1) | JP3027770B2 (en) |
| CA (1) | CA2184386C (en) |
| FI (1) | FI114341B (en) |
| WO (1) | WO1995023801A1 (en) |
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| DE4434093A1 (en) * | 1994-09-23 | 1996-03-28 | Boehringer Mannheim Gmbh | Method for the qualitative and / or quantitative detection of a substance to be determined |
| IL114149A0 (en) * | 1995-06-14 | 1995-10-31 | Yeda Res & Dev | Modified avidin and streptavidin molecules and use thereof |
| FI100276B (en) * | 1996-02-06 | 1997-10-31 | Orion Yhtymae Oy | Method for non-competitive determination of analytes |
| DE19724787A1 (en) * | 1997-06-06 | 1998-12-10 | Biotez Berlin Buch Gmbh Bioche | Streptavidin / avidin coated surfaces |
| ATE225511T1 (en) * | 1997-12-11 | 2002-10-15 | Roche Diagnostics Gmbh | RESOLUTION OF DIAGNOSTIC PROCEDURES USING PEPTIDES FROM D-AMINO ACIDS |
| US6410692B2 (en) * | 1998-02-02 | 2002-06-25 | Novadx, Inc. | Removal of abundant interfering proteins from a liquid sample using a collapsible affinity matrix |
| EP0957360B1 (en) * | 1998-05-06 | 2002-07-31 | Roche Diagnostics GmbH | Removal of disturbances by rheumatoid factors |
| JP3345401B2 (en) * | 1998-08-25 | 2002-11-18 | ユニバーシティ オブ ワシントン | Rapid quantitative analysis of proteins or protein functions in complex mixtures |
| US6143507A (en) * | 1998-10-29 | 2000-11-07 | Boehringer Ingelheim Pharmaceuticals, Inc. | High throughput compatible assay for receptor-TRAF interactions |
| US6629040B1 (en) | 1999-03-19 | 2003-09-30 | University Of Washington | Isotope distribution encoded tags for protein identification |
| WO2001096869A1 (en) | 2000-06-12 | 2001-12-20 | University Of Washington | Selective labeling and isolation of phosphopeptides and applications to proteome analysis |
| US7771922B2 (en) * | 2002-05-03 | 2010-08-10 | Kimberly-Clark Worldwide, Inc. | Biomolecule diagnostic device |
| US7223368B2 (en) * | 2002-05-03 | 2007-05-29 | Kimberly-Clark Worldwide, Inc. | Diffraction-based diagnostic devices |
| US7485453B2 (en) * | 2002-05-03 | 2009-02-03 | Kimberly-Clark Worldwide, Inc. | Diffraction-based diagnostic devices |
| US7214530B2 (en) * | 2002-05-03 | 2007-05-08 | Kimberly-Clark Worldwide, Inc. | Biomolecule diagnostic devices and method for producing biomolecule diagnostic devices |
| US7118855B2 (en) * | 2002-05-03 | 2006-10-10 | Kimberly-Clark Worldwide, Inc. | Diffraction-based diagnostic devices |
| US7223534B2 (en) * | 2002-05-03 | 2007-05-29 | Kimberly-Clark Worldwide, Inc. | Diffraction-based diagnostic devices |
| US7091049B2 (en) * | 2002-06-26 | 2006-08-15 | Kimberly-Clark Worldwide, Inc. | Enhanced diffraction-based biosensor devices |
| US7169550B2 (en) * | 2002-09-26 | 2007-01-30 | Kimberly-Clark Worldwide, Inc. | Diffraction-based diagnostic devices |
| US20050112586A1 (en) | 2003-11-24 | 2005-05-26 | Roland Janzen | Method and composition for stabilizing liquid reagents |
| US20060114172A1 (en) * | 2004-11-26 | 2006-06-01 | Giotti, Inc. | Method and apparatus for LED based modular display |
| US8669052B2 (en) | 2008-06-10 | 2014-03-11 | Rapid Pathogen Screening, Inc. | Lateral flow nucleic acid detector |
| US8445293B2 (en) | 2005-02-09 | 2013-05-21 | Rapid Pathogen Screening, Inc. | Method to increase specificity and/or accuracy of lateral flow immunoassays |
| US8815609B2 (en) | 2008-05-20 | 2014-08-26 | Rapid Pathogen Screening, Inc. | Multiplanar lateral flow assay with diverting zone |
| US9068981B2 (en) | 2009-12-04 | 2015-06-30 | Rapid Pathogen Screening, Inc. | Lateral flow assays with time delayed components |
| US20130196310A1 (en) | 2008-05-20 | 2013-08-01 | Rapid Pathogen Screening, Inc. | Method and Device for Combined Detection of Viral and Bacterial Infections |
| US8609433B2 (en) * | 2009-12-04 | 2013-12-17 | Rapid Pathogen Screening, Inc. | Multiplanar lateral flow assay with sample compressor |
| US8962260B2 (en) | 2008-05-20 | 2015-02-24 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
| US20110086359A1 (en) | 2008-06-10 | 2011-04-14 | Rapid Pathogen Screening, Inc. | Lateral flow assays |
| EP2728354B1 (en) | 2011-06-29 | 2018-06-13 | LSI Medience Corporation | Non-specific reaction inhibitor, method for inhibiting non-specific reaction, and kit |
| WO2013018836A1 (en) * | 2011-08-01 | 2013-02-07 | 日本たばこ産業株式会社 | Method for inhibiting non-specific binding in step of detecting substance in biological sample, and agent for use in the method |
| US10808287B2 (en) | 2015-10-23 | 2020-10-20 | Rapid Pathogen Screening, Inc. | Methods and devices for accurate diagnosis of infections |
| US20210325378A1 (en) * | 2018-09-25 | 2021-10-21 | Siemens Healthcare Diagnostics Inc. | Methods and compositions for removing biotin interference from assays using cyclodextrin traps |
| CN112129933B (en) * | 2019-06-25 | 2024-01-05 | 迈克生物股份有限公司 | Reagent, kit and method for resisting biological interference in immunoassay system |
| CN112129955B (en) * | 2019-06-25 | 2023-04-07 | 迈克生物股份有限公司 | Testosterone detection kit |
| EP4290235A1 (en) * | 2022-06-10 | 2023-12-13 | Siemens Healthcare Diagnostics Products GmbH | Method for neutralizing a biotin interference in binding assays |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8314523D0 (en) * | 1983-05-25 | 1983-06-29 | Lowe C R | Diagnostic device |
| EP0155854B1 (en) * | 1984-03-22 | 1990-09-26 | Bresatec Limited | Non-radioactive biological probes |
| WO1986002077A1 (en) * | 1984-10-02 | 1986-04-10 | Meade Harry M | Production of streptavidin-like polypeptides |
| US4839293A (en) * | 1986-02-24 | 1989-06-13 | The Trustees Of Columbia University In The City Of New York | DNA encoding streptavidin, streptavidin produced therefrom, fused polypeptides which include amino acid sequences present in streptavidin and uses thereof |
| DE3800644A1 (en) * | 1988-01-12 | 1989-07-20 | Boehringer Mannheim Gmbh | PROCESS FOR HIGHLY SPECIFIC DETECTION OF NUCLEIC ACIDS IN SOLUTION |
| US5268306A (en) * | 1988-02-29 | 1993-12-07 | Boehringer Mannheim Gmbh | Preparation of a solid phase matrix containing a bound specific binding pair |
| US4914040A (en) * | 1988-03-03 | 1990-04-03 | Boehringer Mannheim Gmbh | Reagent and method for determination of a polyvalent substance using an immunoaggregate |
| DE3915135A1 (en) * | 1989-05-09 | 1990-11-15 | Boehringer Mannheim Gmbh | PROCESS FOR DETECTING SPECIFICALLY BINDERABLE SUBSTANCES IN KOERPERFLUESSIGKEITEN |
| US5252466A (en) * | 1989-05-19 | 1993-10-12 | Biotechnology Research And Development Corporation | Fusion proteins having a site for in vivo post-translation modification and methods of making and purifying them |
| US5252743A (en) * | 1989-11-13 | 1993-10-12 | Affymax Technologies N.V. | Spatially-addressable immobilization of anti-ligands on surfaces |
| DE4040669A1 (en) * | 1990-12-19 | 1992-06-25 | Behringwerke Ag | USE OF PEPTIDE COUPLES WITH EXTREMELY HIGH SPECIFIC AFFINITY TOGETHER IN THE AREA OF IN VITRO DIAGNOSTICS |
| WO1992022854A1 (en) * | 1991-06-17 | 1992-12-23 | Seiko Epson Corporation | Heat developing apparatus |
| US5474796A (en) * | 1991-09-04 | 1995-12-12 | Protogene Laboratories, Inc. | Method and apparatus for conducting an array of chemical reactions on a support surface |
| DE4135543A1 (en) * | 1991-10-28 | 1993-04-29 | Boehringer Mannheim Gmbh | RECOMBINANT CORE STREPTAVIDINE |
| US5260004A (en) * | 1991-12-02 | 1993-11-09 | The United States Of America As Represented By The Secretary Of The Army | Process of making Langmuir-Blodgett films having photo-electronic properties |
| US5487975A (en) * | 1993-11-15 | 1996-01-30 | Ventana Medical Systems, Inc. | Biotin/avidin formulation |
-
1995
- 1995-03-03 CA CA002184386A patent/CA2184386C/en not_active Expired - Lifetime
- 1995-03-03 WO PCT/EP1995/000776 patent/WO1995023801A1/en active IP Right Grant
- 1995-03-03 US US08/700,435 patent/US5863740A/en not_active Expired - Lifetime
- 1995-03-03 JP JP7522705A patent/JP3027770B2/en not_active Expired - Lifetime
-
1996
- 1996-09-04 FI FI963461A patent/FI114341B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| JPH09510289A (en) | 1997-10-14 |
| FI963461A0 (en) | 1996-09-04 |
| US5863740A (en) | 1999-01-26 |
| WO1995023801A1 (en) | 1995-09-08 |
| CA2184386A1 (en) | 1995-09-08 |
| FI963461A (en) | 1996-09-04 |
| JP3027770B2 (en) | 2000-04-04 |
| FI114341B (en) | 2004-09-30 |
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